Abstract A fundamental function of the kidney is the recovery of filtered water, electrolytes, and proteins in order to conserve valuable nutrients while discarding the final urine. The sequential recovery of electrolytes and water is well understood, however, less is known about the capture of proteins from the filtrate. Protein capture is mediated by two enormous proteins called megalin and cubilin. Despite the critical function of these two molecules, little is known about their molecular mechanisms, and fundamental questions about megalin and cubilin function remain unanswered: How does a single receptor recognize and bind so many different proteins? What is the receptor:ligand stoichiometry and affinity? Do different types of proteins bind to the same receptor molecule at the same time, or do ligands cooperate or compete with one another for binding? These questions have remained unanswered in part because of the large sizes of megalin and cubilin, 600kDa and 450kDa respectively, making their biochemical, molecular and structural analysis daunting. In a labor-intensive undertaking, Drs Shapiro and Brasch and Drs Barasch and Beenken have purified to homogeneity these massive proteins as well as a native megalin-cubilin-albumin complex. The isolated proteins demonstrated non- aggregated, well-behaved single particle behavior in electron microscopy experiments. 3D reconstructions from negative stain EM reveal a remarkable architecture, in which the domains of megalin fold to form a large globular structure in which deep crevices and holes of different sizes are formed by association of the numerous megalin sub-domains. These crevices and holes are large enough to dock different urinary ligands such as NGAL and albumin. Based on these observations, we propose that megalin may act as a ?sponge? with binding pockets complementary to different ligands. The megalin-cubilin-albumin complex appears larger and has distinct structural features. Beginning with these preliminary data, our goal is to define the structure of the megalin and megalin-cubilin protein-recycling receptors, and their complexes with filtered-protein ligands. We will first use single particle cryo-EM to assign the identities of receptor sub-domains visualized in 3D EM reconstructions, and use these assignments to identify ligand-interacting regions of the receptors. To achieve high resolution, some of these studies will be performed with smaller recombinant receptor fragments with structure determination by x-ray crystallography. We will assess the function of different receptor domains with mutagenesis and analysis of ligand binding by SPR, using both known megalin and cubilin ligands as well as novel candidates isolated from urine of humans with defined Donnai Barrow mutations. This work is the first to visualize full-length megalin and megalin-cubilin structures; we expect that our structures will connect the function of the giant recycling receptors to sequence and chemistry, and we expect this will be transformative for kidney biology.